Studies on tubulin, the principal protein of microtubules, will be directed along two lines. One, efforts will be made to extend the resolution in three-dimensional structural studies beyond the 20 A level recently achieved by electron microscopy and image reconstruction on sheets of tubulin formed in the presence of zinc. These efforts will combine low dosage procedures on unstained samples with new computational methods involving a real-space cross-correlation approach designed to overcome curvature in the lattice lines of the sheets which have limited resolution by conventional Fourier reconstruction methods. Two, efforts will be made to clarify the organization of tubulin units in microtubules. Investigations which have revealed a mixed lattice for microtubules reassembled in vitro, with some inter-protofilament contacts involving like subunits and others involving unlike subunits, will be extended to the structure of microtubules in vivo by purifying microtubules without the usual cycles of dissociation and reassembly. Related investigations will be carried out to reveal the absolute structural polarity of microtubules initiated from various microtubule organizing centers, such as centrioles and kinetochores, as well as flagellar axonemes. These studies would complement the conclusions reached by recent kinetic analyses of initiation. An aspect of the proposed research related to both the zinc-induced sheets and the structure of microtubules concerns the identification of the individual alpha and beta subunits of tubulin. Although two types of morphological units can be resolved alternating along protofilaments, it is not possible to identify which is alpha and which is beta. Specific labelling experiments are proposed to aid in this identification, as well as efforts to isolate the subunits in a form that can be fully reconstituted. This latter approach would facilitate labelling and also permit a number of other questions to be answered on the roles of the individual subunits.